Project Summary Bacteria constantly adapt to their environment and selective pressure through genetic mutations. They also mutate in response to vaccine selective pressure, leading to the rise of vaccine-escape strains and loss of vaccine efficacy. To avoid this, we propose that using proteins essential for bacterial growth and virulence is more advantageous to develop vaccine antigens with decreased propensity to lead to vaccine-escape strains. To identify these proteins, we developed a novel dual-RNA sequencing methodology and characterized bacterial transcriptomes during infection to identify proteins important for bacterial growth, virulence, and survival. We showed that genes encoding proteins involved in iron and heme acquisition in Bordetella pertussis are the most highly up-regulated genes during murine respiratory infection. These proteins are required for growth and virulence, surface-exposed, and highly conserved. In previous studies, we showed that proteins involved in iron acquisition provide protection against the respiratory pathogen Pseudomonas aeruginosa in mice. The overall hypothesis of this proposal is that proteins involved in bacterial iron acquisition are ideal antigens for inclusion in acellular vaccines against a wide spectrum of bacterial pathogens. This hypothesis is based on our preliminary data and on the evidence that iron is an essential micronutrient required for growth and pathogenesis. This hypothesis will be tested on B. pertussis, the causative agent of whooping cough, a disease on the rise and re-emerging as a major public health concern in the US and around the world. The rise of vaccine-escape strains is one of the main contributing factors to the loss of protection provided by currently available acellular pertussis vaccines (aP). We propose that the high expression levels of the surface exposed iron-acquisition proteins, together with their requirement for growth and virulence, will lead to an increase in overall vaccine protection, and a reduced propensity to generate vaccine-escape strains. The objective of this application is to evaluate the protection conferred by B. pertussis iron-acquisition proteins to improve the efficacy of aPs and slow vaccine-driven strain evolution. We will generate antigens from B. pertussis iron and heme acquisition receptors as peptides or using innovative detergent-free protein purification methods. We will test them as vaccine antigens against B. pertussis alone, or in combination with aP to determine their antigenicity and efficacy for protection against B. pertussis respiratory infections in mice. At the completion of this project, we expect to have formulated a new acellular pertussis vaccine that provides protection against pertussis while slowing vaccine-driven strain evolution, and established an additional proof of concept for the use of iron-acquisition proteins as vaccine antigens against bacterial pathogens.